CN106690244B - Preparation and application of novel sweet gynostemma pentaphylla sweetener - Google Patents
Preparation and application of novel sweet gynostemma pentaphylla sweetener Download PDFInfo
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- 235000009508 confectionery Nutrition 0.000 title claims abstract description 28
- 235000003599 food sweetener Nutrition 0.000 title claims abstract description 25
- 239000003765 sweetening agent Substances 0.000 title claims abstract description 25
- 235000002956 Gynostemma pentaphyllum Nutrition 0.000 title claims description 43
- 240000006509 Gynostemma pentaphyllum Species 0.000 title claims description 39
- 238000002360 preparation method Methods 0.000 title claims description 4
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- 238000000034 method Methods 0.000 claims abstract description 17
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- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 claims abstract description 5
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- 238000003756 stirring Methods 0.000 claims description 8
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
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- UYRUBYNTXSDKQT-UHFFFAOYSA-N glycyrrhizic acid Natural products CC1(C)C(CCC2(C)C1CCC3(C)C2C(=O)C=C4C5CC(C)(CCC5(C)CCC34C)C(=O)O)OC6OC(C(O)C(O)C6OC7OC(O)C(O)C(O)C7C(=O)O)C(=O)O UYRUBYNTXSDKQT-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines Containing Plant Substances (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
A sweetener extracted from sweet herba Gynostemmatis is prepared by removing chlorophyll, fat and saponin from sweet herba Gynostemmatis tea with organic solvent, extracting with distilled water at 70 ~ 100 deg.C for 0.5H ~ 2H, extracting with H2O2Decolorizing, deproteinizing by Sevage method, dialyzing in distilled water with dialysis bag with cut-off molecular weight range of 500 ~ to remove macromolecular polysaccharide and protein impurities, dialyzing in distilled water with dialysis bag with cut-off molecular weight of 100 ~ to remove a small amount of salt and organic solvent.
Description
Technical Field
The invention belongs to the technical field of natural product extraction, and particularly relates to a structural representation and application of a preparation method for extracting, separating and purifying a sweetener from sweet gynostemma pentaphylla.
Background
The gynostemma pentaphylla is also called heaven grass, foenum-graecum herb, super ginseng, radix cirsii leinii, radix polygoni multiflori, fiveleaf gynostemma herb and the like, and the Latin literature name is as follows: gynostemma pentaphylum (Thunb.) Makino Cucurbitaceae, Gynostemma, herbaceous climbing plant, thin and weak stem, branch, longitudinal edge and groove, and no hair or short and soft hair. Japanese called sweet vine tea. Gynostemma pentaphylla likes the climate of yin-dampness and mildness, and is mostly wild in the shade places such as under the forest and the brook, and the gynostemma pentaphylla climbs the herbaceous plants for many years. The gynostemma pentaphylla medicine is mainly distributed in Pinli, Gansukang county, Hunan and Hubei provinces, Yunnan, Guangxi provinces and other provinces in Shaanxi province, is called 'southern ginseng', has higher medicinal content of gynostemma pentaphylla growing in south, is called as magical 'long-life herb' in folk, and has the effects of reducing blood fat, regulating blood pressure, preventing and treating thrombus, preventing and treating cardiovascular diseases, regulating blood sugar, promoting sleep, delaying senescence, preventing and treating cancers, improving immunity, regulating human physiological functions and the like. In 1986, the national ministry of sciences listed gynostemma pentaphylla as the first place of famous and precious Chinese medicinal materials to be developed in the star fire project, and listed in the list of health care products by the national ministry of health in 3.5.2002. The new variety of the gynostemma pentaphylla, namely 'en wu ye honey' and 'en qiye sweet', obtains the second-class prize of scientific and technological progress of Hubei province in 2008, and contains novel natural non-sugar sweet substances which are not contained in other varieties of the gynostemma pentaphylla and other plants.
At present, gynostemma pentaphylla is mainly used as a tea beverage, and because the gynostemma pentaphylla contains a plurality of components such as saponin, aspartame, protein, polysaccharide and the like, and the effects of the components are different, the gynostemma pentaphylla is taken as a health-care product for a long time, and some side effects such as nausea, vomiting, abdominal distension, diarrhea (or constipation), dizziness, dim eyesight, tinnitus and the like can be caused to some people. Therefore, the extraction of the effective components is very important for researching the curative effect and the health care effect of the traditional Chinese medicine.
Non-patent literature, "separation and identification of Gynostemma pentaphyllum sweet substance" (Liu jin Long, Sun Dong et al, food science, 2007, 28 (10): 480) 483) reports that ultrasonic wave, ZTC1+1 impurity removing agent, G-10 gel chromatography and other extraction and separation technologies are adopted to obtain the sweet substance, the molecular weight of the sweet substance is 251, the molecular formula of the sweet substance is estimated to be C13H19N2O3, and the molecular structure of the sweet substance is different from that of sucrose, glucose, currently developed fructus momordicae sweet element, glycyrrhizin, ficollin and stevia rebaudiana sweet element. The molecular purification method of the sweet substance of the ' Enqi phyllanthus ' gynostemma pentaphylla is that dried leaf powder of the ' Enqi phyllanthus is degreased with ethyl ether → 95% ethanol is soaked and ultrasonically crushed → hot-dip is extracted → centrifugal filtration → ZTC is decontaminated → centrifugation → concentration → methanol is dissolved in acetone and precipitate is dissolved with deionized water and is extracted with saturated n-butyl alcohol → water phase is concentrated → silica gel column is passed → Sephadex G-10 is purified and freeze-dried. There are mainly 2 components, one with molecular weight of 274, no structure can be determined; another molecular weight is 170, and the molecular formula may be C11H10N 2. However, both of these methods are small extraction in the laboratory, and the molecular purity is not high, and the structure is not completely clarified.
The relevant gynostemma pentaphylla sweetener patent documents do not exist at present.
Disclosure of Invention
The invention provides a method for gradually separating, extracting and purifying the gynostemma pentaphylla sweetener aiming at the problem that the structure of the current gynostemma pentaphylla sweetener is not completely clarified, and simultaneously, a plurality of characterization technologies are applied to track and characterize the purity and the structure of the gynostemma pentaphylla sweetener, so that the gynostemma pentaphylla sweetener with high purity and accurate structure is finally obtained.
The invention also aims to provide the gynostemma pentaphylla sweetener prepared by the method.
The third purpose of the invention is to provide the application of the gynostemma pentaphylla sweetener.
The sweet substance of the sweet gynostemma pentaphylla has the main structure of glucose 6-ethyl aldehydic acid-1, 4-alpha-D galactose, is a natural non-sugar sweetener, has no chemical taste or metal taste, and has sweetness 300 ~ 1200 times that of cane sugar.
In order to achieve the purpose of the invention, the following technical steps are adopted.
1. Selecting sweet herba Gynostemmatis tea, removing chlorophyll, fat and saponin with organic solvent in Soxhlet extractor, and removing solvent from residue in ventilated place.
2. Adding distilled water with the mass of 1-7 times into the gynostemma pentaphylla treated in the step 1, keeping the temperature at 70-100 ℃, stirring and extracting for 0.5-2 h, and repeatedly extracting for 2 times. The supernatants were combined by centrifugation and concentrated in vacuo using a rotary distiller.
3. Adding ammonia water into the concentrated solution obtained in the step 2 to adjust the pH value of the solution to 8 ~ 9, and adding 30% of H in portions2O2Decolorization was 1 ~ 3 days.
4. Deproteinizing by Sevage method, adding chloroform/n-butanol mixed solvent with volume ratio of 5/1, stirring the mixture with a stirrer for 30 min, centrifuging, collecting upper layer water solution, and removing denatured protein at the junction of water layer and solvent layer and lower layer chloroform solution. The deproteinization is repeated for a plurality of times until no free protein is detected at the characteristic absorption peak of 280 nm of the protein by an ultraviolet detector. The aqueous solution was then concentrated under vacuum using a rotary distiller.
5. And (3) dialyzing the concentrated solution after decolorization and protein removal in distilled water for 2 ~ 4 days by using a dialysis bag with the molecular weight cut-off range of 500-2000, changing water at least once every day, taking the dialyzate outside the dialysis bag, and concentrating in vacuum by using a rotary distiller.
6. Dialyzing the solution in the step 5 in distilled water by a dialysis bag with molecular weight of 100-500, taking the solution in the dialysis bag, and concentrating the solution in vacuum by a rotary distiller.
7. And (4) drying the solution in the step 6 by using a freeze dryer to obtain a sweetener sample.
As another simplified technical scheme of the invention, in the above technical steps, step 1, step 3 and step 4 can also be omitted, the sweet gynostemma pentaphylla tea leaves are directly added with water for extracting fat-soluble impurities without removing the fat-soluble impurities in step 1, and the extract is subjected to protein removal in step 5 without decoloring in step 3 or deproteinizing in step 4. The details are as follows.
1. Selecting sweet gynostemma pentaphylla tea, adding distilled water with the mass of 1-7 times, keeping the temperature at 70-100 ℃, stirring and extracting for 0.5-2 h, and repeatedly extracting for 2 times. The supernatants were combined by centrifugation and concentrated in vacuo using a rotary distiller.
2. Dialyzing with 500-1000 molecular weight dialysis bag in distilled water for 2 ~ 4 days, changing water at least once every day, collecting dialysate outside the dialysis bag, and vacuum concentrating with rotary distiller.
3. Dialyzing the solution in the step 2 in distilled water by a dialysis bag with molecular weight of 100-500, taking the solution in the dialysis bag, and concentrating the solution in vacuum by a rotary distiller.
4. And drying by a freeze dryer to obtain the sweetener sample.
Applying liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS) or the like to the prepared sweetener sample,1H, 13C and two-dimensional Nuclear Magnetic Resonance (NMR), infrared spectrum (IR), element analysis and other researches show that the main structure of the aspartame sample is glucose 6-ethyl aldehyde-1, 4-alpha-D galactose.
The invention extracts and separates the high-purity sweetener from the sweet gynostemma pentaphylla, and determines that the chemical structure of the sweetener is mainly glucose 6-ethyl aldehyde-1, 4-alpha-D galactose. The method is simple and easy to implement and has low cost.
Drawings
FIG. 1 sweet Gynostemma pentaphyllum sweetener13C nuclear magnetic resonance image.
FIG. 2 is an infrared spectrum of gynostemma pentaphyllum.
FIG. 3 is a liquid chromatogram-mass spectrum of gynostemma pentaphyllum.
FIG. 4 shows the molecular structure of gynostemma pentaphyllum sweetener.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
1. Selecting sweet gynostemma pentaphylla tea 100g, removing chlorophyll, fat and saponin with organic solvent in a Soxhlet extractor, taking residue, and removing solvent in ventilated place.
2. Adding 500mL of distilled water into the gynostemma pentaphyllum treated in the step 1, keeping the temperature at 70-90 ℃, stirring and extracting for 0.5-2 h, and repeating the extraction for 2 times. The supernatants were combined by centrifugation and concentrated to 200mL in vacuo using a rotary distiller.
3. Adding ammonia water into the concentrated solution in the step 2 to adjust the pH value of the solution to 8 ~ 9, and adding 3mL of 30% H in portions2O2Decolorization was 1 ~ 3 days.
4. Deproteinizing by Sevage method, namely adding 20mL of chloroform/n-butanol mixed solvent with volume ratio of 5/1, stirring the mixture with a stirrer for 30 min, centrifuging, taking the upper aqueous solution, removing denatured protein at the junction of the aqueous layer and the solvent layer and the lower chloroform solution, repeatedly deproteinizing for many times until no free protein is detected at 280 nm of characteristic absorption peak of protein by an ultraviolet detector, taking the aqueous solution, and vacuum-concentrating by using a rotary distiller at the temperature of lower than 70 ℃ and under the vacuum degree of 0.005 ~ 0.05.05 MPa.
5. And (3) dialyzing the concentrated solution after decolorization and protein removal in distilled water for 2 ~ 4 days by using a dialysis bag with the molecular weight cut-off range of 500-1000, changing water at least once every day, taking the dialyzate outside the dialysis bag, and performing vacuum concentration by using a rotary distiller under the same conditions.
6. Dialyzing the solution in the step 5 in distilled water by using a dialysis bag with molecular weight of 100-500, taking the solution in the dialysis bag, and carrying out vacuum concentration by using a rotary distiller under the same conditions.
7. Freeze drying the concentrated solution in a freeze dryer at a temperature below-20 deg.C and a vacuum degree of 0.001 ~ 0.005.005 MPa to obtain the sweet taste essence sample.
The infrared spectrum analysis of the sample shows that the characteristic peak of the sample is 619 cm-1，1078cm-1，2934 cm-1，3409cm-1And 1384 cm-1And 1596 cm-1It should be a polyhydroxylated saccharide compound (FIG. 1). sample elemental analysis is shown in Table 1, the H: C: O ratio is close to 2: 1: 1, indicating a carbohydrate, the H: C ratio is slightly less than 2, indicating the presence of an unsaturated olefin, NMR 1H shows a very strong hydroxyl proton peak at 4.70ppm, amplified to show the presence of saturated groups-OCH- (3.0 ~ 4.3.3 ppm), -CH2(1.2~1.7ppm)、-CH3(0.7ppm) protons and the like, mainly polyhydroxy compounds, and a nuclear magnetic resonance 13C spectrum shows that a sample spectrum peak is sharp, mainly small molecular oligosaccharide (57 ~ 100 ppm) contains methyl (13ppm), methylene (25, 29ppm) and double bond (126,135 ppm) (figure 2). liquid chromatography-mass spectrometry shows that ultraviolet chromatogram detection mainly has one peak, contains a small amount of impurities and has the purity of 95%, positive ion mass spectrometry shows that the mass-to-nucleus ratio of molecular ions is 385 (figure 3), and a gas chromatography mass spectrometry analysis mainly has the structure of glucose 6-ethyl aldehyde-1, 4-alpha-D galactose after methylation derivatization (figure 4).
TABLE 1 results of elemental analysis of samples
| Sample (I) | C | H | O，% | H:C (mol/mol) | H:O (mol/mol) |
| Example 1 | 41.90 | 6.14 | 47.42 | 1.75 | 2.07 |
| Example 2 | 41.79 | 6.71 | 51.5 | 1.93 | 2.08 |
Example 2
1. Selecting 100g sweet gynostemma pentaphylla tea, adding 500mL distilled water, keeping the temperature at 70-80 ℃, stirring and extracting for 0.5-2 h, and repeatedly extracting for 2 times. The supernatants were combined by centrifugation and concentrated to 200mL in vacuo using a rotary distiller.
2. Dialyzing with 500-1000 molecular weight cut-off dialysis bag in distilled water for 2 ~ 4 days, changing water at least once every day, collecting dialysate outside the dialysis bag, and vacuum concentrating with rotary distiller at temperature below 70 deg.C and vacuum degree of 0.005 ~ 0.05.05 MPa.
3. Dialyzing the solution in the step 2 in distilled water by a dialysis bag with molecular weight of 100-500, taking the solution in the dialysis bag, and concentrating the solution in vacuum by using a rotary distiller under the same conditions.
4. Freeze drying the concentrated solution in a freeze dryer at a temperature below-20 deg.C and a vacuum degree of 0.001 ~ 0.005.005 MPa to obtain the sweet taste essence sample.
The infrared spectrum analysis of the sample shows that the characteristic peak of the sample is polyhydroxy carbohydrate. Elemental analysis of the samples is shown in Table 1, with a H: C: O ratio of approximately 2: 1: 1, indicated as carbohydrate, with a ratio of H to C of slightly less than 2, indicating the presence of unsaturated olefins.
Claims (8)
1. A sweetener extracted from sweet Gynostemma pentaphyllum Makino with molecular formula of C14H24O12The molecular weight is 384, the molecular structure is glucose 6-ethyl aldehyde-1, 4-alpha-D galactose, and the preparation of the novel sweetener comprises the following technical steps:
(1) selecting sweet gynostemma pentaphylla tea, removing fat-soluble impurities chlorophyll, fat and saponin by using an organic solvent in a Soxhlet extractor, taking residues in a ventilated place, and removing the solvent;
(2) adding distilled water 1 ~ 7 times the mass of the treated gynostemma pentaphyllum, keeping the temperature at 70 ℃ and ~ 100 ℃ and stirring for extraction for 0.5h ~ 2h, repeating the extraction for 2 times, centrifuging, filtering, mixing the supernatant, and performing vacuum concentration by using a rotary distiller;
(3) adding ammonia water into the concentrated solution obtained in the step 2 to adjust the pH value of the solution to 8 ~ 9, and adding 30% of H in portions2O2Decolorizing for 1 ~ 3 days;
(4) deproteinizing by Sevage method, namely adding a chloroform/n-butanol mixed solvent with a volume ratio of 5/1, stirring the mixture for 30 min by using a stirrer, centrifuging, taking an upper aqueous solution, separating denatured protein at the junction of a water layer and a solvent layer and a lower chloroform solution, repeatedly deproteinizing for many times until no free protein is detected at 280 nm of a characteristic absorption peak of the protein by an ultraviolet detector, and taking the aqueous solution and then vacuum-concentrating by using a rotary distiller;
(5) dialyzing the concentrated solution after decolorization and protein removal in distilled water for 2 ~ 4 days by using a dialysis bag with the cut-off molecular weight range of 500 ~ 2000, changing water at least once every day to remove macromolecular polysaccharide impurities and protein, taking the dialyzate outside the dialysis bag, and performing vacuum concentration by using a rotary distiller;
(6) dialyzing the solution obtained in step 5 in distilled water with dialysis bag with cut-off molecular weight of 100 ~ 500, removing small amount of salt and organic solvent, collecting the solution in dialysis bag, and vacuum concentrating with rotary distiller;
(7) and (4) drying the solution in the step 6 by using a freeze dryer to obtain a sweetener sample.
2. The method of claim 1, wherein: the organic solvent for removing chlorophyll, fat and saponin is one or more of acetone, ethyl acetate or petroleum ether.
3. The method of claim 1, wherein the rotary evaporator is operated at a temperature of less than 70 ℃ and a vacuum of 0.005 ~ 0.05.05 MPa.
4. The method of claim 1, wherein: h2O2The decolorization is carried out at normal temperature and pressure, H2O2Mixing with extractive solution at volume ratio of 1: 100 ~ 1: 50, adding H2O2Before the reaction, ammonia water is added to adjust the pH value of the solution to 8 ~ 9.
5. The method of claim 1, wherein the ratio of the Sevage reagent to the extract is 1: 20 ~ 1: 5 by volume when deproteinizing by the Sevage method.
6. The method of claim 1, wherein the freeze-drying temperature is-20 ℃ or lower and the vacuum degree is 0.001 ~ 0.005.005 MPa.
7. The method of claim 1, wherein: another method for preparing the sweetener is to omit the steps 1, 3 and 4, directly extract the sweet gynostemma pentaphylla tea leaves from the step 2 by adding water, remove protein from the step 5, remove a small amount of salt and organic solvent from the step 6, and dry the sweet taste tea leaves in the step 7 to obtain a sweetener sample.
8. Use of the gynostemma pentaphyllum sweetener according to claim 1, wherein: can be used as flavoring beverage and food additive.
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